Starches from grain, cereals, and tubers, e.g., cornstarch, are widely used in the industrial manufacture of products such as sugar syrups and biofuels. For example, high fructose corn syrup (HFCS) is a processed form of corn glucose syrup having high fructose content and a sweetness comparable to sucrose, making HFCS useful as a sugar substitute in soft drinks and other processed foods. HFCS production currently represents a billion dollar industry. Similarly, the production of ethanol from starches is a rapidly expanding industry.
Syrups and biofuels can be produced from starch by an enzymatic process that catalyzes the breakdown of starch into glucose. This enzymatic process typically involves a sequence of enzyme-catalyzed reactions:
(1) Liquefaction: Alpha-amylases (EC 3.2.1.1) first catalyze the degradation of a starch suspension, which may contain 30-40% w/w dry solids (ds), to maltodextrans. Alpha-amylases are endohydrolases that catalyze the random cleavage of internal α-1, 4-D-glucosidic bonds. Because liquefaction typically is conducted at high temperatures, e.g., 90-100° C., thermostable alpha-amylases, such as alpha-amylases from Bacillus sp., are preferred for this step. Alpha-amylases currently used for this step, e.g., alpha-amylases from B. licheniformis, B. amyloliquefaciens, and Geobacillus stearothermophilus (AmyS), do not produce significant amounts of glucose. Instead, the resulting liquefact has a low dextrose equivalent (DE), containing maltose and sugars with high degrees of polymerization (DPn).
(2) Saccharification: Glucoamylases catalyze the hydrolysis of alpha-1,4-glucosidic linkages of maltodextrins formed after liquefaction from non-reducing ends, releasing D-glucose. Saccharification produces high glucose syrup. Debranching enzymes, such as pullulanases, can aid saccharification.
(3) Further processing: A branch point in the process occurs after the production of a glucose-rich syrup. If the final desired product is a biofuel, yeast can ferment the glucose-rich syrup to ethanol. On the other hand, if the final desired product is a fructose-rich syrup, glucose isomerase can catalyze the conversion of the glucose-rich syrup to fructose.

Alpha-amylases are isolated from a wide variety of bacterial, fungal, plant, and animal sources. Many industrially important alpha-amylases are isolated from Bacillus sp., in part because of the generally high capacity of Bacillus to secrete amylases into the growth medium. In addition, Bacillus alpha-amylase variants with altered while more desirable properties are obtained through genetic engineering. Furthermore, there is a need for blends of alpha-amylases, or variants thereof, which can capitalize on the best properties of at least two alpha-amylases of different origins.
The Fuelzyme®-LF alpha-amylase (SEQ ID NO: 2)(Verenium Corp.) is an engineered alpha-amylase obtained through DNA shuffling of three parental enzymes. See Richardson et al., J. Biol. Chem. 277: 26501-26507 (2002); U.S. Pat. No. 7,323,336. The advantageous properties of the Fuelzyme®-LF alpha-amylase include: effective viscosity reduction at a lower dose, improved thermostability, and broad pH operating ranges. The use of this alpha-amylase, however, is currently limited to biofuel applications, e.g., ethanol production, because it results in ineffectual glucose syrup that is not suitable for downstream applications such as sweetener applications. Specifically, saccharification of starch liquefact from Fuelzyme®-LF alpha-amylase results in iodine-positive saccharide (IPS), which indicates incomplete starch hydrolysis. Thus, if a way could be found to fully exploit the advantages of the Fuelzyme®-LF alpha-amylase in starch processing, particularly in sweetener applications, by using an optimized blend of alpha-amylases, this would also represent a useful contribution to the art.